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Introduction

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells (1-3) with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.

GIBCO® media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout™ DMEM supplemented with KnockOut™ Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture (3-9). The launch of KnockOut™ SR XenoFree, based on the formulation of KnockOut™ SR but using human-derived or human recombinant proteins under cGMP conditions, allows the transition of pluripotent stem cell research to the clinic. This gold standard media system can now be used for feeder-free culture with the addition of KnockOut™ SR Growth Factor Cocktail.

Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the < conditions. Invitrogen has developed Knockout™ SR Growth Factor Cocktail to allow you to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout™ SR XenoFree.

Just before pre-equilibrating the complete medium to temperature and gases, aseptically add the required volume of 2-mercaptoethanol (55 mM stock concentration) for a 0.1 mM final concentration. For example, to prepare 100 mL of KSR-FF medium add 182 μL of 55 mM 2-mercaptoethanol (1:550 dilution) Alternatively, the 2-mercaptoethanol may be added to the 1X completed medium and stored at 2–8°C for up to one week.

Adapting Human iPSCs to KSR XenoFree FF Medium

Culture the human iPSCs on human foreskin fibroblasts or MEF feeder cells until they are 70–80% confluent.

Pre-warm the required volume of TrypLE™ in a 37 °C water bath. Refer to Table 1 below for details on the volumes required.

Pre-equilibrate the required volume of KSR XenoFree FF in a 37°C water bath for15 min. Refer to Table 1 below for details on the volumes required.

Aspirate the medium from the culture dishes, and add an appropriate amount of TrypLE™ . Incubate the dishes at 37°C for 3-5 minutes.

Aspirate the TrypLE™ from each culture vessel and wash off the MEF feeder cells gently with D-PBS (2 to 3 times).

Add an appropriate amount of complete KSR XenoFree FF medium to each culture vessel. Use a cell scraper or a 5 mL pipette to gently scrape the cells off the surface of culture vessel.

Collect the cell suspension from each culture dish into separate 15 mL conical tubes. Rinse each culture vessel with an appropriate amount of complete KSR XenoFree FF medium, and add the D-PBS rinse medium into the 15 mL conical tubes containing the cell suspension. Be cautious not to break the cell clumps into single cells

Aspirate the supernatant from the iPSC pellet. Resuspend the pellet in an appropriate amount of KSR XenoFree FF medium according to the split ratio (Table 1). Do not break the cell clumps to a smaller size, because the smaller clumps do not attach well to the surface.

Note: We recommend a split ratio of 1:2 for the first 3 passages after the iPSCs have been passaged directly from the iPSC feeder-based culture medium to KSR XenoFree FF medium. Normally, a split ratio between 1:3 and 1:5 is appropriate, but passaging at 1:2 ensures the higher density of cells needed when adapting into a feeder-free culture.

Aspirate the CELLstart™ solution from the pre-coated culture vessel, and slowly add an appropriate amount of cell suspension to each culture vessel.

Move the culture dish back and forth and side to side several times to disperse the cells across the surface of the dish. Gently place the culture dish in a 37°C incubator with a humidified atmosphere of 4 to 6% CO2 in air. Replace the spent medium with KSR XenoFree FF every day.

Passaging human iPS cells using KSR XenoFree FF

Observe the human iPSCs growing in complete KSR XenoFree FF under the microscope to confirm that the cells are 70–80% confluent and ready to be subcultured. Refer to Figure 1.

Remove the vessel from the incubator, aspirate the TrypLE™, and gently wash the cells with D-PBS.

Gently scrape the cells off the surface of the culture dish using a cell scraper, and transfer the cells to a sterile 15mL centrifuge tube.

Rinse the culture dish twice with KSR XenoFree FF, gently “spraying off” any cells that have not detached. Pool the rinse medium with the cells in the 15mL tube.

Centrifuge the tube at 200 × g for 5 minutes at room temperature to pellet the cells.

Carefully aspirate the supernatant without disturbing the cell pellet and discard it.

Gently flick the tube to fully dislodge the cell pellet from the tube bottom.

Gently resuspend the cells in pre-equilibrated KSR XenoFree FF using a 5mL serological pipette. Do not triturate. Note: it is critical at the step to gently resuspend the cells without using force to avoid damage.

Transfer the cells to a fresh 60-mm CELLstart™-coated dish at the desired split ratio and move the culture dish back and forth and side to side several times to disperse the cells across its surface.

Place the culture dish in a 37°C incubator with a humidified atmosphere of 4 to 6% CO2 in air.

The next day, gently replace the spent medium with KSR XenoFree FF to remove cell debris. Replace the spent medium everyday thereafter. Observe the iPS cells daily and passage them as needed (approximately every 4–5 days). Passaging is recommended when the cells reach 70-80% confluence. For iPS cell cryopreservation and thawing, refer to our protocol titled “Cryopreserving and Recovering Human iPSCs in Complete KnockOut™ Serum Replacement XenoFree with or without feeders”.

Note: If iPS cell lines are extremely problematic, a further level of caution can be taken by maintaining a culture in each prior passage medium while starting the next level of adaptation. For example, when passaging the 25/75 MEF-conditioned medium / KSR XenoFree FF culture (as described above), iPS cells can be passaged into both 100% KSR XenoFree FF AND 25/75 medium. If the 100% culture does poorly, adaptation can be resumed using the backup 25/75 culture.